FI85214C - Process for the preparation of liposomes and packaging for use in the preparation of a liposome aerosol - Google Patents

Description

1 85214

The present invention relates to a process for the preparation of liposomes from a lipid substance and water capable of forming a liposome aerosol. This method produces liposomes In sltu using a pressurized aerosol system.

Certain therapeutically active compounds are administered to patients by inhalation therapy, i.e., they are administered to the airways by nasal or oral administration in the form of steam or gas or mist together with a carrier in the form of steam or gas. Likewise, in certain cases, in the treatment of the skin, for example in the treatment of open wounds or sprains, in the treatment of skin conditions or for cosmetic purposes, it may be desirable to administer the active compound by spraying as a gas or vapor or mist. In all cases, it is desirable that as much of the active compound in good condition as possible extend to the intended site of action, and sometimes that the active compound then remain in good condition for some time at the intended site of action. It can be said that the active compound should be administered in such a way that it does not degrade during transport or immediately upon contact with the patient.

Thus, for example, when administering a bronchodilator compound known as bitolter (disclosed in GB Patent No. 1,298,771) as an asthma medication, it is desirable to administer the compound in such a way as to obtain maximal bronchial permeability, thereby detecting the most effective levels of the active compound at the required sites.

It has previously been proposed to prepare liposome compositions containing biologically active compounds, and such compositions are disclosed, for example, in GB Patent Publication Nos. 1,575,343, 2,852,145,575,344 and 2,013,609 A. However, in such known compositions, the liposome material is preformed and dried. typically by lyophilization (freeze-drying). The liposome material is then re-usable by mixing with water.

Aerosol compositions containing a variety of cosmetic and therapeutic agents are also known - see, for example, GB Patent Nos. 780,885, 993,702, 1,302,671, 1,381,184, 1,516,195 and 2,001,334 A. Although such known aerosol compositions may contain lipid ingredients, these ingredients are invariably present only as dispersing, suspending, emulsifying or similar substances. In addition, such compositions are intended to produce either a dry spray so that no liposome formation is possible or, as in the case of the ointment in Patent No. 780,885, they are included as an emulsified premix of all lipids and water.

In addition, as disclosed in U.S. Patent No. 3,594,476,20, the preparation of lecithin aerosols useful in the treatment of lung diseases has been proposed, which aerosols may optionally contain other therapeutic agents such as antibiotics. In the present preparation method, a suspension of DL-dipalmitoyl-α-le-sitin in water (or saline) is first prepared, in which the drug is dissolved in water or saline, which suspension is then nebulized, i. converted into an aerosol in an ultrasonic generator, which was provided with a flow of air acting as a carrier. Thus, the presented method is dependent on a relatively expensive ultrasonic device for the preparation of the necessary ready-made mixture of lecithin and water, as well as when using the initial mixture containing lecithin suspended in water. The ultrasound device may also be noisy and may not be suitable for use.

3,85214

In addition, as disclosed in U.S. Patent No. 4,371,451, it has been proposed to prepare lecithin-based compositions for lubricating the surface of cooking vessels containing water, lecithin and dimethyl ether as a propellant. Since, according to this patent, the solutions are released from the aerosol container, the disclosed method is again dependent on the use of an initial mixture in which lecithin is dispersed in water. In addition, what has been sought is rapid drying and a thin layer of lesin-10 that does not foam, in contrast to liposomals when used as such.

We have now surprisingly found that by using a pressurized aerosol system, the liposome material can be instantly formed from separate aqueous and lipid components, and that the various active agents can be administered more effectively by spray administration with a liposome carrier formed in situ upon spray administration. Such a liposome agent formed by in situ administration of a spray mixture is also useful per se, for example in the treatment of lung diseases, as is known in the art.

Accordingly, the present invention relates to a process for the preparation of liposomes, which process is characterized in that at least two separate components 25 are brought together under pressure, the first component containing water and the second component containing a lipid substance, and that the mixture is fed under pressure through a nozzle or other device. a liposome and an aerosol spray containing.

The method of the invention provides for the in situ or sequential preparation of liposomes using a pressurized aerosol system. In the invention, the first water-containing component and the second component containing a dry lipid substance are mixed to give a spray which may contain liposomes as such or as a liposome carrier for one or more other substances. Thus, in a preferred embodiment of the invention, a liposome aerosol is prepared comprising a liposome carrier and a separate active agent, which is generally biologically active and / or useful in treating or treating the human or animal body. Such an active agent is also generally a non-lipid-like agent, although in some cases a lipid may be used to transfer the active moiety, e.g., in association therewith.

Alternatively, the invention may be used in the preparation and provision of a liposome aerosol as such, without a separate active compound or other substance. Such aerosols could have utility in a number of ways, either in the preparation of liposomes for various uses or in therapy. In particular, they could be useful in childbirth where newborns with respiratory distress (respiratory distress syndrome) could be helped to breathe more normally by administering a lipid to their bronchial region to increase the low amount of naturally occurring lung surfactant found in children.

Preferably, however, a separate active agent is provided which may initially be present in either the first or, more preferably, the second component. Such active substance may be present as a suspension prepared, for example, from a dry powder, in the first or second component, but is preferably in solution in one of the components. By using a solution of the active substance, it is possible to avoid subsidence, as would be the case in the case of suspension, and the obvious variation in the dosage level due to subsidence.

In the method according to the invention, the formation and / or spraying of the mixture results in the preparation of the liposome substance. It is believed that the formation of the mixture results in an emulsion consisting of lipid micelles suspended in water, from which liposomes are then formed upon spraying. In addition, when the active agent is present, the liposome agent contains the active agent mediated by an aqueous medium, either enclosed in encapsulated form between the lipid layers or as a sandwich structure between the lipid molecules. By varying the conditions under which the two components are mixed and / or sprayed, the size of the liposomes and the relative proportions of free and bound active agent 10 can be varied within wide limits, the latter always up to about 100% incorporation. Thus, for example, by forming and / or spraying an aerosol mixture at varying agitation rates, for example as obtained with turbulence conditions obtained, for example, in a mixing chamber and / or connected feeders and / or spray nozzle, the spray can be formed with either a high proportion of free a substance - which gives a rapid effect with its slow-release active substance reservoir -20 or one with less free active substance - which gives a slower effect with its extended and controlled release of the active substance. Thus, in the latter case, a composition containing the active agent may be prepared in which a greater proportion of the agent may be delivered in a "protected" form to the intended site of action and / or otherwise acting in a sustained release manner.

The method of the invention is useful in preparing, in a form suitable for immediate administration, compositions containing various biologically active agents and / or other agents useful in the treatment or treatment of the human or animal body. Typically, the method can be used to prepare compositions with therapeutically. 35 active compounds, including those used in 6 85214 veterinary treatments, as well as biologically active reagents, cosmetically active substances, compounds having a nutritional value and other substances used for the treatment or treatment of the human or animal body.

Suitable cosmetically active substances include products for skin and hair care, for example moisturizers, artificial tanning agents (optionally in combination with colorants), water-soluble flame retardants, antiperspirants, deodorants, astringents and tonics, tints, scars, scars, depilatory products, perfumes, animal or vegetable tissue extracts, water-soluble hair dyes, anti-dandruff agents, anti-sebum agents, oxidizers, 15 e.g. bleaches and reducing agents.

Therapeutically active compounds that may be mentioned include vitamins, steroids, hormones, or prickly peptides, enzymes, vaccines, anti-inflammatory agents, antibiotics and bactericides. However, the method of the invention is particularly suitable for the administration of therapeutically active compounds used in inhalation therapy, such as bronchodilators and asthma medications, as well as antitumor agents. Such compounds, either in or with a liposome carrier, are capable of penetrating the bronchial branches, thus allowing the efficacy of the treatment to be advantageously varied. Similarly, in the treatment of skin wounds or other dermatological or cosmetic treatments, a liposome carrier may be used to produce a (promoting) transforming effect, for example a sustained release effect, thus allowing a certain level of treatment to be maintained for a long time.

In accordance with the foregoing, and by way of example only, the following specific compounds may be mentioned and an -____: 35 states: 7 85214

Active compound Type of compound Method of administration

Stanozole Anabolic Steroid Topically

Hydrocortisone (and Anti-Inflammatory Topical Esters) Steroid 5 Betamethasone (and Topical Anti-Inflammatory Esters, e.g. Steroid Its Valerate)

Bitolterol (and its Catecholamine-Pulmonary Inhalation Esters, e.g. its tube dilator 10 mesylate)

Salbutamol Catecholamine-pulmonary-inhalation-vasodilator

Theophylline Xanthene-bronchodilator Inhalation extender 15 Sodium cromoglyc Non-steroidal asthma Inhalation N-acetylmuramyl Macrophage activator Inhalation L-alanyl-D-isoenzyme (for tumor suppression dipeptide in 20 rim dipeptide)

Propranolol β-blocker and fruit Oral / anti-vaginal

Those skilled in the art will, of course, appreciate that many other active agents may be used, as well as other routes of administration. For example, compositions prepared by the method of the invention containing suitable active ingredients may be administered directly to, for example, the eye or even intravenously.

Mention may also be made of biologically active substances which may also be therapeutically active, such as vaccines or other biologically active substances which are useful as reagents, such as antigens and / or antibodies, e.g. anti-rabbit IgG. Such reagents may be useful in the so-called as markers 8 85214 e.g. in testing blood samples, typically for possible disease.

The liposome agent prepared in the invention may be positively or negatively charged or may be neutral. The substance contains a particulate lipid substance which is in aqueous dispersion or suspension and may contain one or more active substances. In addition, the lipid substance may be in the form of unilamellar and / or multilamellar lipid bilayers, which may be associated with any of the other active substances mentioned.

The liposome agent typically comprises a phospholipid and is preferably a phospholipid containing phosphatidylcholine, e.g. diplamitoylphosphatidylcholine as such or in the form of lecithin; phosphatidylethanolamine; 15 or phosphatidylserine. The liposome agent may be formed from the phospholipid alone or in combination with cholesterol and / or at least one compound that may provide a charge to the liposome agent. Thus, for example, the liposome may be charged using diacetyl phosphate or phosphatidic diacid, which may give a negatively charged lipid, or stearylamine, which may give a positively charged lipid.

The use of the above substances and their proportions, as well as the wide range of variations and variations in the ratios that can be used, are well known to those skilled in the art. However, by way of example only, in a preferred embodiment of the invention, the second component may contain a phospholipid consisting of phosphatidylcholine and cholesterol, typically in relative molar proportions of about 8: about 1 to about 2, e.g. about 8.5: 1.

In another preferred embodiment of the invention, the second component may comprise a phospholipid consisting of phosphatidylcholine, cholesterol, and disethyl phosphate, typically in molar ratios of about 8: about 1 to 9,852,14 to about 2: about 1 to about 0.05, e.g., about 8.5 : l: about 0.7 to about 0.07.

In another preferred embodiment of the invention, the second component may comprise a phospholipid consisting of phosphatidylcholine, cholesterol and phosphatidic acid, typically in relative molar proportions of about 8: about 1 to about 2: about 1 to about 0.1, e.g. about 8.5 : 1: about 0.1.

In yet another preferred embodiment of the invention, the second component may contain a phospholipid consisting of phosphatidylcholine, cholesterol, and stearylamine, typically in relative molar proportions of about 8: about 1 to about 2: about 1 to about 0.05, e.g., about 8, 5: 1: about 0.06.

Alternatively, the second component may contain, for example: a mixture of phosphatidylcholine, e.g. lecithin and phosphatidic acid, e.g. in a ratio of about 60: about 1; or a mixture of phosphatidylcholine, e.g. lecithin 20 and disethyl phosphate, e.g. in a ratio of about 15: about 1.

The liposome agent prepared in the invention should preferably have a pH in the physiological range, and thus preferably in the range of about 6.8 to about 7.4, more preferably about 7 to about 7.4. Therefore, it may also contain a buffer or buffer system to the extent necessary to obtain and maintain the desired pH, and a phosphate buffer, which may typically contain sodium dihydrogen phosphate and disodium hydrogen phosphate, is very suitable for this purpose. Further, such a buffer may preferably have an aqueous first component. However, in some cases, e.g. when the active substance contains a reagent such as a tracer to be used in the experiment, the pH may be outside the physiological range.

Depending on the ingredients of the second component, it may be necessary or advantageous to include a physiologically acceptable liquid excipient, preferably one in which both the lipid agent and any other active agent are soluble. In this respect, preferred liquid excipients are relatively volatile organic solvents, for example ethanol, chloroform (according to local rules), ether and isopropyl alcohol.

In addition, the first component may also contain a physiologically acceptable liquid excipient in addition to water. Such an excipient may be a volatile organic solvent, preferably a water-miscible organic solvent, of which ethanol is preferred.

In a preferred embodiment of the invention, the first and second components are mixed under pressure using one or more aerosol propellants, which may be an ingredient of one or both components and / or used separately. Preferably, however, the propellant is a component of the second component and the pressure is applied to the first component by means of the propellant kept separate from it.

Typically, one or each propellant may comprise a hydrocarbon or a halogenated hydrocarbon or a mixture of such hydrocarbons. Examples of such propellants are propane, butane, dimethyl ether and Propellant 11 (trichlorofluoromethane), although dichlorodifluoromethane or dichlorotetrafluoroethane or mixtures thereof are preferred. First, the propellant may be formed as a low pressure propellant that develops pressures ranging from about 25 to about 30 psi (about 172-207 kPa), such as a 20/80 weight ratio mixture of dichlorodifluoromethane and dichlorotetrafluoroethane, or the propellant may be a relatively high pressure propellant. which develops pressures up to say 70 psi (about 483 kPa), such as an 80:20 by weight mixture of dichlorodifluoromethane and dichlorotetrafluoroethane. However, it will be appreciated that relative dosage amounts, such as between the 11 8521 4 components, may be monitored and varied e.g. depending on the propellant pressure used for each component.

In the method according to the invention, it is preferred that the first and second components are used separately, that all components are fed in controlled separate doses to the mixing chamber under their own propellant pressure, and that the mixture thus formed is removed from the mixing chamber through a nozzle under at least one propellant pressure. Preferably, the first and second components are also mixed in such a way as to give a measured dose of one of said active ingredients.

Even more preferably, the second component is fed as a metered dose to the metered dose of the first component and is preferably contacted with at least a major portion of the first component in a range of said metered doses. Thus, even more preferably, the method of the invention comprises the following steps in succession, namely: the first component is introduced into the mixing chamber in a measured amount; the measured amount of the second component is introduced into the mixing chamber in a manner that produces turbulent mixing of the two components, preferably by introducing the second component into the mixing chamber at a plurality of locations; and the mixture is removed from the mixing chamber.

The measured amount of the first component is conveniently monitored by placing the first component in the mixing chamber in an amount that fills the chamber and selecting the volume of the chamber to provide the desired measured amount of the first component.

The invention further relates to a kit for use in the manufacture of a liposome aerosol, the kit comprising at least a first and a second chamber, the second ____:35 or both chambers and / or the third chamber containing a propellant material, characterized in that the first chamber contains a first component , with water, and the second chamber contains a second component having a liposome capable of forming a liposome, and that the package comprises means for spraying a mixture of the first and second components from their respective chambers under pressure developed by one or with more propellants.

The first component containing water may be a buffered aqueous substance and the second component in the second chamber is a lipid substance, one of said components being optionally containing, preferably as a suspension or solution, an active ingredient, e.g. for use in treating or treating the human or animal body, the second or both chambers and / or the third chamber contain a propellant.

Preferably, the removal means is such that the mixing ratio of the components is monitored (say at a measured level or a narrow measured range of ratios) so that the desired metered dose of said active substance can be removed from the package.

It should be reiterated that it is preferred that the active agent be in suspension or solution in another component comprising a lipid agent, and more preferably, the active agent is in solution to avoid settling. In general, the active agent, the first and second components, and the propellant are as described above in connection with the process of this invention.

Preferably, the package according to the invention has an outlet mixing chamber and a valve device by means of which the first and second components are introduced from their respective chambers into the mixing chamber under pressure developed by one or more propellants and monitoring the ratio of 35 components (say measured level or i3 8521 4 within a small measured range). a metered dose of the mixture, i.e. the active substance together with the liposome carrier or the liposome itself, can be administered through a removal nozzle.

Preferably, said valve device comprises means (say two valves) for successively introducing the first component to fill the mixing chamber, the measured dose of the second component being introduced into the mixture! into the spray chamber so that turbulent mixing of the two components occurs and the mixture is removed through an outlet nozzle. Preferably, the valve device also comprises means for introducing the second component into the mixing chamber at a plurality of locations within the chamber.

If desired, an additional mixing device can also be used either upstream or downstream of the mixing chamber to provide thorough and / or turbulent mixing conditions to promote emulsion formation prior to removal through the discharge nozzle. Thus, for example, the discharge nozzle system may itself comprise an auxiliary mixing chamber into which the mixture is fed so as to provide new turbulent conditions, for example by tangentially feeding the mixture into the chamber through one or more feed points before removal by the discharge nozzles.

In the package according to the invention, the first comb. The mio may be a first aerosol container or bag, the second chamber may be a second aerosol container or bag, and the containers or other first and second closed chambers may be located: Endically with corresponding valve devices and mixing chambers located between or above the containers or other chambers;

Laterally with corresponding valve devices and mixing chambers located between or above the tanks or other chambers; or 35 The first inside (or vice versa) with the valve devices and mixing chambers located inside or above the outer tank or outer chamber. Aerosol containers using such an arrangement are disclosed, for example, in U.S. Patent Nos. 3,325,056 and 3,326,416.

In the package according to the invention, an outer jacket can be used, in which the closed chambers can be placed and through which the valve device can be arranged. In such a package, it is preferred that the second component contains a propellant and is placed in an inner rigid-walled closed chamber, the first component is placed in an outer flexible-walled closed chamber that preferably surrounds the inner chamber, the jacket is a rigid-walled jacket and the space is surrounded by an outer closed chamber. . A particularly preferred two-component packaging device having these features is that disclosed in EP Patent Application No. 84-10-4589.1 and FI Patent Application No. 843,249, which are based on CH Patent Application No. 4450/83.

However, if desired, the inner chamber may be similar to the outer chamber, i.e., the inner chamber is made of a flexible-walled chamber surrounded by a rigid-walled chamber containing a propellant.

As will be appreciated from the foregoing description, in practicing the present invention, the dosage level of active agent used in each operation of the kit described above may vary widely depending on various factors, in particular: 1. The concentration of active agent in the first and / or second component; and 30 2. The mixing ratio of the first and second components.

In the case of the mixing ratio of the components, it depends on the magnitude of the measured amounts of the first and second components used for mixing, which in turn depends on the particular mixing and / or valve equipment used. In the particular case of the above-mentioned particularly preferred apparatus, the volume of the mixing chamber is preferably about 50 to about 100, e.g. 75 to 100 microliters, and the valve controlling the amount of the second component preferably delivers about 50 to about 100 microliters per operation to the mixing chamber.

With respect to the size of liposomes, we believe that size is primarily regulated by the pressure exerted by the viscosity of the propellant or propellants and / or components. In general, the more components are mixed, the smaller the size of the liposomes. However, the size of the valve outlet and delivery nozzle used to release the mixed components can also affect the size of the liposomes produced. Preferably, such a discharge nozzle has a size of about 350 to about 450 microns.

On the other hand, once the above parameters have been arranged, the dose level obtained can be easily ascertained, as can the amount of any active substance content and the size of the liposomes prepared.

As will be appreciated by those skilled in the art of preparing active agents with carriers, the amount of active agent content in the liposome carrier in this invention may have said effect on the base dose level selected for the total composition. According to it, such a basic dose level is a matter of choice of composition, 25 which depends on the criteria taken into account in advance in any particular composition.

The following examples illustrate the method and aerosol package of the invention.

In the specific examples given, the majority of the compositions contain ammonium molybdate as an electron-dense substance, which was added as a tracer alone to facilitate the detection of liposomes and their diameter measurement by electron microscopy. Those skilled in the art of pharmaceutical compositions will, of course, appreciate that ammonium molyb-. The date, which is quite toxic, should not be part of the actual active composition as administered to the patient. As can be seen when comparing Example 19 and Example 20 below, the omission of ammonium molybdate has no detrimental effect on the amount of active substance content and in fact the amount of content 5 is slightly higher in Example 20. According to this, any exemplary composition can omit molybdate label without disadvantage and the results given can be obtained without it.

Also, because different amounts of active agent are provided by way of example, and because they in turn provide different amounts of free and associated active agent per action, those skilled in the art will again appreciate that the dosage level and type of composition selected for any formulation will depend on the desired dosage regimen. intended to be achieved, as well as any necessary or desired balance between the unbound and the associated active substance.

For example, assuming that the average person applies an ointment composition to the skin at an average rate of 2.2 to 20 mg / cm 2 and assuming that a single application of the aerosol, i.e. a spray, covers 25 cm 2 of skin, the following comparison can be made between the example and standard compositions:

Composition Application rate 25 _ (micrograms / cm2)

Stanozolol Cream (0.1%) 2.3

Stanozolol aerosol from Example 21 4.0

Hydrocortisone cream (1.0%) 23

Hydrocortisone aerosol from Example 25 4.8 30 Betamethasone ointment (0.1%) 2.5

Betamethasone aerosol from Example 27 0.64

Thus, the active agent may be administered in a total dosage amount of more or less (or even as much) 35 as the known standard amount to produce some desired change in effect, with this change in effect increasing with the degree of incorporation selected.

Similarly, in the case of compositions for inhalation, the total dose may vary depending on the effect required. Thus, for example, as illustrated in Example 31, salbutamol can be effectively administered 125 micrograms of slow release agent and 125 micrograms of free agent per application when compared to 100 micrograms per application from 10 standard dispensers. Further, as illustrated in Examples 29 and 30, respectively, the active agent may be administered in much higher or much lower dosages per application than is standard - 933 micrograms versus 200 micrograms (standard 15 for bitolterol in Example 29, and 200 micrograms for 2 micrograms). mg (standard) for sodium cromoglycate in Example 30 - although in both cases the dosage may be adjusted to the standard level by varying the total amount of active substance present in the second or first 20 components, respectively.

Example 1 240 mg of egg lecithin and 28 mg of cholesterol were dissolved in 4 ml of ethanol and the solution was added to 25 g of a 20:80 by weight mixture of dichlorodifluoromethane and di-: 25-chlorotetrafluoroethane propellant. The mixture was then sealed in a first container to which a valve was connected. 25 ml of 3.3 mM aqueous sodium phosphate solution, pH 7.2, was placed in an aluminum bag, which was then sealed in a separate container equipped with a valve and containing 25 g of the above propellant. The tanks were connected by means of a mixing chamber, and each operation of the valve mechanism released an aliquot of lipid solution and an aliquot of phosphate solution into the mixing chamber and the atmosphere outside it through a narrow nozzle. The resulting aerosol contained 35 multilamellar and unilamellar liposomes with an average diameter of 40-1,000 nm.

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Example 2

The procedure of Example 1 was repeated except that the propellant was a 40:60 mixture (parts by weight) of dichlorodifluoromethane and dichlorotetrafluoroethane and the first tank equipped with a valve contained a mixture of 240 mg of egg lecithin, 28 mg of cholesterol and 4 mg of phosphatidic acid dissolved in 4 ml of ethanol, together with 25 g of propellant.

The obtained equipment could be used to prepare an Aero-10 Sol containing negatively charged multilamellar and unilamellar liposomes with a mean diameter of 40-1000 nm.

Example 3

The procedure of Example 1 was repeated except that the first tank with 15 valves contained a mixture of 240 mg of egg lecithin, 28 mg of cholesterol and 1.6 mg of stearylamine dissolved in 4 ml of ethanol, together with 25 mg by weight of 20:80. a mixture of dichlorodifluoromethane and dichlorotetrafluoroethane propellant.

The obtained equipment could be used to prepare an aerosol containing positively charged multilamellar and unilamellar liposomes with a mean diameter of 40-1000 nm.

25 Example 4

The procedure of Example 1 was repeated, except that the first tank with a valve contained a mixture of 240 mg of egg lecithin and 4 mg of phosphatidic acid dissolved in 4 ml of ethanol, together with 25 g by weight of a 20:80 mixture of dichlorodifluoromethane and diol. -klooritetrafluorietaaniponneainetta.

The resulting device could be used to prepare an aerosol containing negatively charged multilamellar and unilamellar liposomes with a mean diameter of .35 40-1000 nm.

19 8 52 1 4

Example 5

The procedure of Example 1 was repeated except that the propellant gas was a 40:60 mixture (parts by weight) of dichlorodifluoromethane and dichlorotetrafluoroethane and the first tank equipped with a valve contained a mixture of 24 mg dipalmitoylphosphatidylcholine, 2.8 mg cholesteryl, 2.8 mg cholesterol, 2 mg and 2 mg of bitolterol mesylate dissolved in 4 ml of ethanol, together with 25 g of propellant.

The resulting system could be used to prepare an aerosol containing negatively charged multilamellar and unilamellar liposomes with a mean diameter of 40 to 1,000 nm. In addition, more than 50% of the bitolterol mesylate was associated with liposomes.

15 Example 6

The procedure of Example 1 was repeated except that the propellant was a 40:60 mixture (by weight) of dichlorodifluoromethane and dichlorotetrafluoroethane and the first tank with a valve contained a mixture of 240 20 mg of egg lecithin, 28 mg of cholesterol, 2 mg of dimethyl phosphate and 20 mg of bitolterol methylate. dissolved in 4 ml of ethanol, together with 25 g of propellant.

The resulting system could be used to prepare an aerosol containing multilamellar and unilamellar liposomes with a diameter of 40-1000 nm. In addition, more than 50% of the bitolterol mesylate was associated with liposomes.

Example 7 50 mg of egg lecithin was dissolved in ethyl alcohol 30 (96% BP) and made up to 1.0 ml with ethyl alcohol to give a lipid component. The resulting solution was transferred to a glass vessel and 7.0 g of dichlorodifluoromethane was added. The glass vessel was then sealed with a 65 microliter metering valve connected to a 75 microliter mixing chamber intended to receive the lipid component exiting through the metering valve and itself forming part of the second valve system.

The assembly thus formed was placed in a second flexible vessel (tubular bag) containing 40 ml of an ammonium molybdate solution (0.5% by weight in deionized water) to give an aqueous component, this component being in communication with the mixing chamber with the second valve closed. The second vessel was then bent into a canister containing 2.0 g of dichloride-10 fluoromethane propellant in such a way that the second valve system (connected to the bag and mixing chamber) could, when activated itself, also cause the first valve to remove the lipid component into the mixing chamber. providing for the removal of the mixed component-15 pathway from the surrounding canister assembly, e.g., as described in the aforementioned FI Patent Application No. 843 249, which is based on CH Patent Application No. 4450/83. The standard 450 pushbutton with nozzle was then connected to another valve-20 system.

The resulting aerosol can assembly could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 40 to 400 nm.

25 Example 8

The procedure of Example 7 was repeated except that the glass vessel was equipped with a 50 microliter metering valve associated with a 100 microliter mixing chamber. The resulting aerosol can assembly could be used to prepare 30 aerosol sprays containing unilamellar and multilamellar liposomes with diameters of 40-1,000 nm.

Example 9

The procedure of Example 7 was repeated except that the glass vessel was equipped with a 100 microliter metering valve associated with a 100 microliter mixing chamber. The resulting 2i 8521 4 aerosol can assembly could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 50-1,000 nm.

Example 10 60.0 mg of eggestin and 7.0 mg of cholesterol were dissolved in 1.0 ml of ethyl alcohol (96% BP). The resulting solution was transferred to a glass vessel, and 6.0 g by weight of a 20:80 mixture of dichlorodifluoromethane and dichlorotetrafluoroethane was added thereto. The glass vessel was then sealed with a 50 microliter 10 ml metering valve connected to a 100 microliter mixing chamber as in Example 7.

The composition thus obtained was placed in a second, flexible vessel containing 40 ml of 3.3 mM aqueous sodium phosphate solution having a pH of 7.2. Again, as in Example 15 7, the second container was bent into an aerosol canister containing 2.0 g of dichlorodifluoromethane propellant so that the second valve system was able to provide an exit from the surrounding aerosol canister assembly. A standard-operated pushbutton with a 450 μm nozzle was then connected to another valve system.

The resulting aerosol can assembly could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 40-1,000 nm.

25 Examples 11-15

The procedure of Example 10 was repeated using in a glass vessel a solution of the following ingredients dissolved in 1.0 any ethyl alcohol (96% BP), namely:

Example No. 30 11. 60.0 mg of egg lecithin, 1.0 mg of β-phosphatidic acid and 7.0 mg of cholesterol.

12. 60.0 mg of egg lecithin, 0.4 mg of stearylamine and 7.0 mg of cholesterol.

13. 60.0 mg of egg lecithin and 1.0 mg of phosphatidic acid.

22 8 521 4 14. 0.5 mg of bitolterol mesylate, 6.0 mg of dipalmitoylphosphatidylcholine, 0.5 mg of diacetylphosphate and 0.7 mg of cholesterol.

15. 5.0 mg of bitolterol mesylate, 60.0 mg of egg-5 sitin, 0.5 mg of bisethyl phosphate and 7.0 mg of cholesterol.

The resulting aerosol canister assembly in each of Examples 11-15 could be used to deliver an aerosol spray containing unilamellar and multilamellar liposomes 10 with diameters of 40-1,000 nm.

Example 16 100 mg of egg lecithin and 6.0 mg of stanozolol were dissolved in ethyl alcohol (96% BP) and made up to 2.0 ml with ethyl alcohol. The resulting solution was transferred to vessel 1a-15 and 9.0 g of dichlorodifluoromethane was added thereto. The glass vessel was then sealed with a 50 microliter metering valve connected to a 100 microliter mixing chamber as in Example 7.

The composition thus formed was placed inside a second, bent-20 vessel containing 40 ml of ammonium molybdate solution (0.5% by weight in deionized water). Again, as in Example 7, the second container was then bent into an aerosol canister containing 2.0 g of dichlorodifluoromethane propellant so that the second valve system could provide an exit from the surrounding aerosol canister assembly. A standard pushbutton with a 450 μm nozzle was then connected to the second valve system.

The resulting aerosol can assembly could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 40-110 nm. Of the 50.0 micrograms of stanozolol released with each use of the second valve system, 21.6 micrograms were found to be associated with lipo-35s.

23 8 5 21 4

Example 17

The procedure of Example 16 was repeated except that the solution used to fill the glass vessel contained 450 mg of egg lecithin and 60 mg of stanozolol dissolved in ethyl alcohol-5 (96% BP) and made up to 3 ml with ethyl alcohol, 4.5 g of dichlorodifluoromethane was added. into the container and the push button had a 360 μm nozzle.

The resulting aerosol can assembly could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes having diameters of 44-440 nm. Of the 500.0 micrograms of stanozolol released by each operation of the second valve system, 35.4 micrograms were found to be associated with liposomes.

15 Example 18

The procedure of Example 17 was repeated, except that the metering valve was a 100 microliter metering valve and the push button had a 450 μm nozzle.

The resulting aerosol can assembly could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 25-190 nm. Of the 1,000.0 micrograms of stanozolol released each time the second valve system operated, 170.5 micrograms were found to be associated with 25 liposomes.

Example 19

The procedure of Example 16 was repeated except that the solution filling the glass vessel consisted of 900 mg of egg lecithin and 60 mg of stanozolol dissolved in ethyl alcohol (96% BP) and the volume of the solution was made to 3.0 ml with ethyl alcohol, and 4 .5 g of dichlorodifluoromethane was added to the vessel.

The obtained aerosol canister device could be used to prepare an aerosol spray containing unilamellar 35 and multilamellar liposomes with a diameter of 40-24 8 5 21 4 350 nm. Of the 500.0 micrograms of stanozolol released with each actuation of the second valve system, 81.3 micrograms were found to be associated with liposomes.

5 Example 20

The procedure of Example 19 was repeated except that the second vessel contained only 40 ml of deionized water.

Of the 500 micrograms of stanozolol released by each operation of the second valve system, 85.0 10 micrograms were found to be associated with liposomes.

Example 21

The procedure of Example 20 was repeated except that the solution in the beaker contained 12 mg of stanozolol.

The obtained aerosol canister device could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 40-350 nm. Of the 100 micrograms of stanozolol released by each operation of the second valve system, 76.8 micrograms were found to be associated with liposomes.

Example 22

The procedure of Example 21 was repeated except that the second vessel contained 40 mL of a solution of ethyl alcohol (1 ti-25% by volume) in deionized water.

The obtained aerosol canister device could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 30 to 500 nm. Of the 100 micrograms of stanozolol released each time 30 operations of the second valve system, 77.3 micrograms were found to be associated with liposomes.

Example 23

The procedure of Example 21 was repeated except that the second vessel contained 40 ml of a solution of ethyl alcohol (5% v / v) in deionized water.

25 8 521 4

The resulting aerosol canister system could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 30-500 nm. Of the 100 micrograms of stanozolol released by each 5-fold operation of the second valve system, 76.3 micrograms were found to be associated with liposomes.

Example 24

The procedure of Example 21 was repeated except that the second vessel contained 40 ml of a solution of ethyl alcohol (10% by volume) in deionized water.

The resulting aerosol canister system could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 30 to 500 nm. Of the 100 micrograms of stanozolol released each time the system was operated, 66.66 micrograms were found to be associated with liposomes.

Example 25

The procedure of Example 17 was repeated except that the solution placed in the la-20 vessel contained 900 mg of egg lecithin and 14.4 mg of hydrocortisone BP dissolved in ethyl alcohol (96% BP) in a volume of 3.0 ml with ethyl alcohol.

The resulting aerosol canister device could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 44-220 nm. Of the 120 micrograms of hydrocortisone BP released by each actuation of the second valve system, 54.0 micrograms were found to be associated with 30 liposomes.

Example 26

The procedure of Example 16 was repeated except that the solution in the beaker contained 841 mg of egg lecithin, 54.8 mg of diacetyl phosphate and 14.4 mg of hydrocortisone BP dissolved in ethyl alcohol (96% BP) with a solution volume of 26 8 5 21 4 made 3, To 0 ml with ethyl alcohol and 4.5 g of dichlorodifluoromethane were added to the vessel.

Of the 120 micrograms of hydrocortisone 5 BP released at each actuation of the second valve system, 45.6 micrograms were found to be associated with liposomes.

Example 27

The procedure of Example 26 was repeated except that the beaker solution contained 841 mg of egg lecithin and 4.8 10 mg of betamethasone valerate BP / USP / NF dissolved in ethyl alcohol (96% BP) in a volume of 3.0 ml with ethyl alcohol.

The resulting aerosol canister device could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 20-300 nm. In addition, 16 micrograms of betamethasone valerate was released with each operation of the second valve system.

Example 28 The procedure of Example 27 was repeated except that betamethasone was not included in the solution placed in the glass vessel.

The resulting aerosol canister device could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 30-1,000 nm.

Example 29

The procedure of Example 26 was repeated except that the solution in the beaker contained 841 mg of egg lecithin and 112 30 mg of bitolterol mesylate dissolved in ethyl alcohol (96% BP) in a volume of 3.0 ml with ethyl alcohol.

The resulting aerosol canister device could be used to prepare an aerosol spray containing unilamellar 35 and multilamellar liposomes with diameters of 27,821 4 25-250 nm. Of the 933 micrograms of bitolterol mesylate released by each operation of the second valve system, 545 micrograms were found to be associated with liposomes.

5 Example 30

The procedure of Example 28 was repeated except that the second vessel contained 40 ml of a solution of sodium cromoglycate USP XX, BP '80 (2 mg / ml) and ammonium molybdate (5 mg / ml) in 0.1 M sodium phosphate buffer pH 10. 7.4.

The obtained aerosol canister device could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 35-700 nm. Of the 200 micrograms of sodium cromoglycate released each time the system was operated, an amount of 22 micrograms was found to be associated with liposomes.

Example 31

The procedure of Example 18 was repeated except that 60 mg of stanozolol was replaced with 15 mg of salbutamol base 20 BP.

The resulting aerosol canister system could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of 30-300 nm. Of the 250 micrograms of salbutamol released at each 25-fold operation of the second valve system, 125 micrograms were found to be associated with liposomes.

Example 32

The procedure of Example 28 was repeated except that the second vessel contained 10 ml of a solution of N-acetyl-muramyl-L-alanyl-D-isoglutamine [NALAG] (100 micrograms / ml) and ammonium molybdate (5 mg / ml) deionized. in the water.

The resulting aerosol canister assembly could be used to prepare an aerosol spray containing unilamellar 28 8521 4 and multilamellar liposomes. In addition, 10 micrograms of NALAG was released each time the second valve system was operated.

Example 33 5,841 mg of egg lecithin was dissolved in ethyl alcohol (96% BP) and made up to 3.0 ml with ethyl alcohol. The resulting solution was transferred to a glass vessel and 4.5 g of dichlorodifluoromethane was added thereto. The glass vessel was then sealed with a 50 microliter metering valve connected to a 10 to 100 microliter mixing chamber as in Example 7.

The device thus formed was placed in a second, flexible vessel containing 35 ml of dilution (50 microliters / ml) of anti-rabbit IgG (1/64 titer against 1/640 normal rabbit serum as determined by agar-agar block precipitation-15 titration) and ammonium molybdate (5 mg / ml) in deionized water. Again, as in Example 7, the second container was then folded into a canister containing 2.0 g of dichlorodifluoromethane propellant so that the second valve system could cause separation from the entire aerosol canister device. A standard weight button with a 450 μm nozzle was then attached to the second valve system.

The resulting aerosol canister device could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes. Each operation of the second valve system released 5 microliters of anti-rabbit IgG (1/64 titer).

It is to be understood that the invention is not limited to the details of the foregoing specific examples. Thus, for example, many other lipid agents can be used to form the desired liposome agent and can serve as carriers for a wide variety of active agents. Many other Aero solo equipment can also be used, especially one of the other special equipment mentioned above. 35 In addition, the required pressure can be provided in other ways than by using one or more aerosol propellants.

In general, as will be appreciated from the foregoing description, the invention comprises a claimed method for preparing liposomes in situ using a pressurized aerosol system, with or without liposomes or with a separate active ingredient, and a kit for use in such preparation.

10

Claims (19)

A process for preparing liposomes of a lipid material capable of forming a liposome and of water, characterized in that at least two separate components are brought together under pressure, the first component comprising water and the second component a lipid material, and that the mixture is pressurized through a nozzle or some other device, thereby obtaining an aerosol spray containing liposomes. A method according to claim 1, characterized in that at least one of said first and second components includes an active material which is biologically active and / or useful in the treatment or care of the human or animal body, such as a therapeutic or cosmetic. active material. Process according to claim 2, characterized in that the active material is in solution in the first or second component. 20 4. A process according to claim 2 or 3, characterized in that the active material is a bronchodilator, an anti-asthmatic compound, an anti-tumor agent, an anti-inflammatory agent, a contraceptive or an anabolic steroid. 25 5. A method according to claim 2 or 3, characterized in that the active material is an antigen, an antibody or some other biologically active reagent. Method according to any of the preceding claims, characterized in that the lipid material in the second component is a phospholipid material which is selected to form a positively charged, negatively charged or neutral liposome. Method according to any of the preceding claims, characterized in that the second component includes a physiologically acceptable liquid auxiliary and / or the first component includes a physiologically acceptable liquid auxiliary in addition to water. Process according to any of the preceding claims, characterized in that the first and second components are mixed under pressure provided by at least one aerosol propellant, which may constitute an ingredient of one or both components and / or which is provided. separate. 10 9. A process according to claim 8, characterized in that a propellant is an ingredient of the second component and pressure is applied to the first component via a propellant which is separate from it. 15 Method according to any of the preceding patent claims, characterized in that the first and second components are isolated from each other, each component being measured in controlled individual boxes in a mixing chamber under their own propellant pressure, and the thus, the mixture formed is discharged from the mixing chamber via a nozzle under pressure from at least one of said propellants. Method according to any of the preceding patent claims, characterized in that the second component is delivered as a measured dose to a measured dose of the first component. Method according to claim 10 or 11, characterized in that in sequence the first component is supplied to the mixing chamber for filling of the chamber, a measured dose of the second component is delivered to the mixing chamber in such a way that turbulent mixing of the two components The mixture is extracted and the mixture is run from the mixing chamber. A package for use in the preparation of a liposome aerosol, comprising at least one first and a second chamber, wherein one or both chambers and / or a third chamber contains a propellant material, characterized in that the first chamber contains a first component containing water and the second chamber contains a second component containing a lipid material capable of forming a liposome and the package including devices, in which the spray is a mixture of the first and second component flowing from respective chambers under pressure developed by one or more propellants. Packaging according to claim 13, characterized in that at least one of said first and second components is intended to include an active material which is biologically active and / or useful in the treatment or care of the human or animal body, and that the run-out device is such that the mixing ratio of the components can be followed, whereby a desired measured dose of the active material can be run out. Packaging according to claim 13 or 14, characterized in that the package also includes an outlet nozzle and a mixing chamber and valve means with which the first and second components are brought from their respective chambers into the mixing chamber under pressure, which is generated by means of a pressure chamber. or several propellants, and the relationship between the components is monitored, the desired measured dose of the mixture being discharged through the outlet nozzle. Packaging according to claim 15, characterized in that the valve means comprise devices which sequentially allow the first component to be brought into the mixing chamber for filling thereof, then a measured dose of the second component is introduced into the mixing chamber in such a way that both components undergo a turbulent mixture and the mixture is driven through the outlet nozzle. 37 8 521 4 Packaging according to claim 15 or 16, characterized in that the valve means include means for permitting the second component to be brought into the mixing chamber at a plurality of locations within the chamber. Packaging according to any of claims 15-17, characterized in that the first and second chambers are closed chambers, one being arranged inside the other, and that the valve means and the mixing chamber are located in and / or above the outer chamber. chamber. A package according to claim 18, characterized in that the second component includes a propellant and is located in an inner, rigid-walled closed canister, that the first component is located in an outer closed cannon with flexible walls, that the package includes an outer stiff-walled housing, within which the chambers are located and through which the valve means can be operated, and that the space between the outer, closed chamber and the surrounding housing contains a propellant. 20